Blood–brain barrier shuttle peptides are increasingly more potent and versatile tools to enhance drug delivery to the brain.

Homogeneous antibody–drug conjugates (ADCs), generated by site‐specific toxin linkage, show improved therapeutic indices with respect to traditional ADCs. However, current methods to produce site‐specific conjugates suffer from low protein expression, slow reaction kinetics, and low yields, or are limited to particular conjugation sites. Here we describe high yielding expression systems that efficiently incorporate a cyclopropene derivative of lysine (CypK) into antibodies through genetic‐code expansion. We express trastuzumab bearing CypK and conjugate tetrazine derivatives to the antibody. We show that the dihydropyridazine linkage resulting from the conjugation reaction is stable in serum, and generate an ADC bearing monomethyl auristatin E that selectively kills cells expressing a high level of HER2. Our results demonstrate that CypK is a minimal bioorthogonal handle for the rapid production of stable therapeutic protein conjugates.

The high selectivity and affinity of antibody binding have made antibodies all-pervasive tools in therapy, diagnosis, and basic science. A plethora of chemogenetic approaches has been devised to make antibodies responsive to stimuli ranging from light to enzymatic activity, temperature, pH, ions, and effector molecules. Within a single decade, the field of activatable antibodies has yielded marketed therapeutics capable of engaging antigens that could not be targeted with traditional antibodies, as well as new tools to control intracellular protein location and investigate biological processes. Many opportunities remain untapped, waiting for more efficient and generally applicable masking strategies to be developed at the interface between chemistry and biotechnology.

Peptides show high promise in the targeting and intracellular delivery of next-generation bio- and nano-therapeutics. However, the proteolytic susceptibility of peptides is one of the major limitations of their activity in biological environments. Numerous strategies have been devised to chemically enhance the resistance of peptides to proteolysis, ranging from N- and C-termini protection to cyclization, and including backbone modification, incorporation of amino acids with non-canonical side chains and conjugation. Since conjugation of nanocarriers or other cargoes to peptides for targeting and cell penetration may already provide some degree of shielding, the question arises about the relevance of using protease-resistant sequences for these applications. Aiming to answer this question, here we provide a critical review on protease-resistant targeting peptides and cell-penetrating peptides (CPPs). Two main approaches have been used on these classes of peptides: enantio/retro-enantio isomerization and cyclization. On one hand, enantio/retro-enantio isomerization has been shown to provide a clear enhancement in peptide efficiency with respect to parent L-amino acid peptides, especially when applied to peptides for drug delivery to the brain. On the other hand, cyclization also clearly increases peptide transport capacity, although contribution from enhanced protease resistance or affinity is often not dissected. Overall, we conclude that although conjugation often offers some degree of protection to proteolysis in targeting peptides and CPPs, modification of peptide sequences to further enhance protease resistance can greatly increase homing and transport efficiency.

Phage display is a powerful approach for evolving proteins and peptides with new functions, but the properties of the molecules that can be evolved are limited by the chemical diversity encoded. Herein, we report a system for incorporating non‐canonical amino acids (ncAAs) into proteins displayed on phage using the pyrrolysyl‐tRNA synthetase/tRNA pair. We improve the efficiency of ncAA incorporation using an evolved orthogonal ribosome (riboQ1), and encode a cyclopropene‐containing ncAA (CypK) at diverse sites on a displayed single‐chain antibody variable fragment (ScFv), in response to amber and quadruplet codons. CypK and an alkyne‐containing ncAA are incorporated at distinct sites, enabling the double labeling of ScFv with distinct probes, through mutually orthogonal reactions, in a one‐pot procedure. These advances expand the number of functionalities that can be encoded on phage‐displayed proteins and provide a foundation to further expand the scope of phage display applications.

The blood–brain barrier (BBB) hampers the delivery of therapeutic proteins into the brain. BBB-shuttle peptides have been conjugated to therapeutic payloads to increase the permeability of these molecules. However, most BBB-shuttles have several limitations, such as a lack of resistance to proteases and low effectiveness in transporting large biomolecules. We have previously reported on the THRre peptide as a protease-resistant BBB-shuttle that is able to increase the transport of fluorophores and quantum dots in vivo. In this work, we have evaluated the capacity of linear and branched THRre to increase the permeability of proteins in cellular models of the BBB. With this purpose, we have covalently attached peptides with one or two copies of the BBB-shuttle to proteins in order to develop chemically well-defined peptide–protein conjugates. While THRre does not enhance the uptake and transport of a model protein in BBB cellular models, branched THRre peptides displaying two copies of the BBB-shuttle result in a 2.6-fold increase.